gD基因插入增强gI/gE/gM缺失重组伪狂犬病病毒对小鼠的免疫效果

doi:10.11843/j.issn.0366-6964.2026.01.028

摘要/Abstract

摘要：

本研究旨在构建双拷贝gD基因的重组伪狂犬病病毒rPRV-AH-gI^-/gE^-/gM^-/gD⁺，并对其免疫效力进行评价。以rPRV-AH-gI^-/gE^-为亲本毒株，利用同源重组技术，缺失gM基因并在其缺失位置插入PRV gD基因表达盒，构建重组病毒rPRV-AH-gI^-/gE^-/gM^-/gD⁺。用10^7.0 TCID₅₀ 的rPRV-AH-gI^-/gE^-、rPRV-AH-gI^-/gE^-/gM^-、rPRV-AH-gI^-/gE^-/gM^-/gD⁺制备灭活疫苗，免疫4周龄昆明小鼠2次，间隔3周，并设商品化疫苗和DMEM对照组，免疫后定期采血，通过中和试验检测小鼠血清中和抗体水平，ELISA试验检测小鼠血清总抗体水平，在首免后6周用10^5.5 TCID₅₀ PRV-AH进行攻毒试验。结果显示：rPRV-AH-gI^-/gE^-/gM^-/gD⁺中插入的gD基因获得了表达，表达蛋白大小约为53 ku；rPRV-AH-gI^-/gE^-/gM^-/gD⁺组小鼠在首免后4、6周中和抗体及血清总IgG抗体水平均显著高于rPRV-AH-gI^-/gE^-、rPRV-AH-gI^-/gE^-/gM^-组（P<0.01），但与商品化疫苗组相比差异不显著（P>0.05）；用PRV-AH攻毒后，rPRV-AH-gI^-/gE^-/gM^-/gD⁺、rPRV-AH-gI^-/gE^-/gM^-、rPRV-AH-gI^-/gE^-和商品化疫苗组的攻毒保护率分别为100%、70%、60%和90%。综上表明，gD基因的插入可以提高重组病毒的免疫保护效果，为PRV流行毒株候选疫苗的研制奠定了一定的基础。

关键词: 伪狂犬病病毒, 双拷贝gD, 同源重组, 免疫效力

Abstract:

In this study， we aimed to develop recombinant PRV rPRV-AH-gI^-/gE^-/gM^-/gD⁺ with double gD and evaluate its immune efficacy. The recombinant virus rPRV-AH-gI^-/gE^-/gM^-/gD⁺ was constructed by deleting gM gene and inserting gD expression cassette at the deficient region. Four-week-old Kunming mice were immunized twice with 10^7.0 TCID₅₀ inactivated rPRV-AH-gI^-/gE^-， rPRV-AH-gI^-/gE^-/gM^-， rPRV-AH-gI^-/gE^-/gM^-/gD⁺ at an interval of 3 weeks. Commercial vaccine and DMEM were as the control group. After immunization， regular blood was collected， neutralizing antibody levels in sera were detected by neutralization test， and total antibody levels were detected by ELISA. At 6 weeks of first immunization， mice were challenged with 10^5.5TCID₅₀ PRV-AH. The inserted gD gene was expressed with the molecular weight of 53 ku in the rPRV-AH-gI^-/gE^-/gM^-/gD⁺. The neutralizing and total IgG antibody levels of rPRV-AH-gI^-/gE^-/gM^-/gD⁺ group were significantly higher than those of rPRV-AH-gI^-/gE and rPRV-AH-gI^-/gE^-/gM^- at 4 and 6 weeks after first immunization （P<0.01）. However， compared with the commercial vaccine group， the difference was not significant （P>0.05）. After challenged with PRV-AH， the protection rates were 100%， 70%， 60% and 90% of the rPRV-AH-gI^-/gE^-/gM^-/gD⁺， rPRV-AH-gI^-/gE^-/gM^-， rPRV-AH-gI^-/gE^- and commercial vaccine groups respectively. The results showed that the insertion of gD gene can improve the immune protection effect of the recombinant virus which is helpful in developing vaccine candidate for PRV control.

Key words: pseudorabies virus, double gD, homologous recombination, immune efficacy

中图分类号:

S852.659.1

陆灵光, 刘家齐, 卢佩琪, 吴瑞彬, 黄丹妮, 刘佳悦, 梁婉琪, 琚春梅. gD基因插入增强gI/gE/gM缺失重组伪狂犬病病毒对小鼠的免疫效果[J]. 畜牧兽医学报, 2026, 57(1): 327-336.

LU Lingguang, LIU Jiaqi, LU Peiqi, WU Ruibin, HUANG Danni, LIU Jiayue, LIANG Wanqi, JU Chunmei. Immune Effect of gD Gene Insertion-enhanced gI/gE/gM Deleted Recombinant Pseudorabies Virus in Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2026, 57(1): 327-336.

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引物名称 Gene name	引物序列（5′→3′） Primer sequences
gM-LA1-F	CATCGTTCCCGTCTGGTGTC
gM-LA1-R	TTCGTCGAGGTCGTCGTGTC
gM-RA1-F	AGAGGCTGATGTGGAAGCTGTT
gM-RA1-R	TGATAGAGGTCTGCGGGTTCG
Sosoo-EGFP-F	ACCTGCAGGCATGCAAGCTTCGTTACATAACTTACGGTAAAT
Sosoo-EGFP-R	CCACATCAGCCTCTAAGCTTTAAGATACATTGATGAGTTTGG
Sosoo-gD-F	GCAGAAATCGTCGACCTGCAGGCATGCAAGCTTCAGATATACGCGTTGAC
Sosoo-gD-R	TCCTGGCCAACAGCTTCAACATCAGCCTCTACCTCAGAAG CCATAGAGC